Modifications of antimetabolite gemcitabine for incorporation in CpG oligonucleotides

ABSTRACT

This Divisional application of patent application Ser. No. 10/768,996, entitled “Novel Oligonucleotides And Related Compounds” discloses a class of chemical compounds which have been demonstrated to possess cancer fighting properties. The parent application disclosed oligonucleotides for selectively killing cancerous cells over noncancerous cells by incorporating and covalently linking antimetabolite prodrugs via CpG moieties, for the anitmetabolite Gemcitabine and other compounds with known cancer fighting properties. This application discloses modifications of Gemcitabine for incorporation into CpG oligonucleotides for improved biochemical and biological properties.

CROSS REFERENCE TO RELATED APPLICATIONS

This application for patent is a Divisional application of patentapplication Ser. No. 10/768,996, entitled “Novel Oligonucleotides andRelated Compounds” filed on Jan. 30, 2004, by Suresh C. Srivastava,Satya P. Bajpai and Kwok-Hung Sit. This Divisional application is beingfiled as a result of a restriction requirement in the parentapplication. This application contains no new matter and claims priorityfrom its parent, application Ser. No. 10/768,996, the entire contents ofwhich application are incorporated herein by reference.

FIELD OF THE INVENTION

This application relates to a class of chemical compounds which havebeen demonstrated to possess cancer fighting properties. It wasdetermined during the prosecution of the parent application that theoriginally filed claims 38-44 which were directed to chemicalmodification of the antimetabolite Gemcitabine belonged in class 536,subclass 4.1. This Divisional application pertains to those claims inclass 536, subclass 4.1.

BACKGROUND OF THE INVENTION

In the parent application it was disclosed that the antimetaboliteprodrug of Gemcitabine possesses strong anti cancer properties providedit is covalently linked to one of the nucleotides of theoligonucletoides having CpG motifs wherein the antimetabolite prodrug islinked by a 3′-3′ linkage, a 5′-5′ linkage, a 3′-5′ linkage, or a 5′-3′linkage. This invention is sometimes referred in the present applicationas the “prior” invention.

A large number of prior art references were given in the parentapplication as filed and during its prosecution to specify thebackground for the parent application. The present applicationrecapitulates the segments relevant to the present application.

The studies of Dr. Kwok-Hung Sit and colleagues (Yee-Jiun Kok, MyintSwe, and Kwok-Hung Sit, Biochemical and Biophysical Communications, 294934-939, 2002), suggest a relationship between the cell death andimmunostimulatory activity. With the effective ODN-CpG binding, there isstrong inhibition of CpG DNA fragmentation, resulting in site specificresistance to cleavage, and thereby prevent necrosis and apoptosis. CpGoligonucleotides referred in the previous publication, however,independently seem to enhance the immunostimulatory activity. On theother hand inhibition of megabase fragmentation of the highly conservedGCn*GC motifs by complementary ODN's will help to protect complementaryCpG DNA from degradation. Our approach as disclosed in the priorapplication for ODN design and incorporation of potent anticancer drugis a novel approach and presents enormous future potential in molecularmedicine. The incorporated anticancer drug can be cleaved by one of theseveral endolytic cleavage mechanisms. This should result in hydrolysisof a phosphodiester bond, esterase hydrolysis of the ester linkagesoutlined in the details of claims or amidate hydrolysis will liberatethe anticancer drug. While the CpG ODN will act as complementary DNA-ODNconjugate for the stability from degradation, it also seems that withproper design selection of the CpGn*CpG ODN, immunostimulatoryproperties of the ODN could be available within the cell.

The hydrolysis of Gemcitabine (or another prodrug) which is attached viaan ester linkage to a CpG oligo, is envisaged to be easily hydrolyzed byintracellular esterases (Ghosh, M and Mitra, A. K., Pharm. Res., 8,771-775, 1009).

It was observed that the sugar and bases of one or more nucleotides thatmake up the oligonucleotide may have one or more substitutions andexamples of one or more nucleotides having a 2′-o-alkyl, 2′-n-alkyl, or2′-halogen modifications on the sugar. Preferably the alkyl is a C1-C6alkyl were described.

It was observed that the sugar and bases of one or more nucleotides thatmake up the oligonucleotides may include one or more protecting groupsfor stability during oligonucleotide synthesis or in vivo conditions.

It was further noted that oligonucleotides can be obtained from existingnucleic acid sources (e.g.

genomic or cDNA), but are preferably synthetic, and have a definedsequence (e.g. produced by oligonucleotide synthesis).

The delivery vehicles were discussed in detail. It was noted that an“oligonucleotide delivery complex” is an oligonucleotide associated with(e.g. ionically or covalently bound to; or encapsulated within) atargeting means (e.g. a molecule that results in a higher affinitybinding to a target cell, such as that of a B-cell or natural killer(NK) cell, and/or increased cellular uptake by target cells). Examplesof oligonucleotide delivery complexes include oligonucleotidesassociated with: a sterol (e.g. cholesterol), a lipid (e.g. cationiclipid, virosome or liposome), or a target cell specific binding agent(e.g. a ligand recognized by a target cell specific receptor). Preferredcomplexes must be sufficiently stable in vivo to prevent significantuncoupling prior to internalization by the target cell. However, thecomplex should be cleavable or otherwise accessible under appropriateconditions within the cell so that the oligonucleotide is functional.(Gursel, J. Immunol. 167: 3324, 2001.)

“Pharmaceutically acceptable carriers” that were shown to be useful inthe prior invention are conventional; see for example, Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 15th Edition (1975), which is incorporated herein by reference.

It was noted that in general, the nature of the carrier will depend onthe particular mode of administration being employed. For instance,parenteral formulations usually comprise injectable fluids that includepharmaceutically and physiologically acceptable fluids such as water,physiological saline, balanced salt solutions, aqueous dextrose,glycerol or the like as a vehicle. For solid compositions (e.g., powder,pill, tablet, or capsule forms), conventional non-toxic solid carrierscan include, for example, pharmaceutical grades of mannitol, lactose,starch, or magnesium stearate. In addition to biologically-neutralcarriers, pharmaceutical compositions to be administered can containminor amounts of non-toxic auxiliary substances, such as wetting oremulsifying agents, preservatives, and pH buffering agents and the like,such as sodium acetate or sorbitan monolaurate.

It was also discussed that the oligonucleotides of the invention can beformulated for intratracheal administration or for inhalation. Suchcompositions can take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and can contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Other pharmacologicalexcipients are known in the art.

A prefered, pharmaceutically acceptable carrier is lipofectin.

For therapeutic or prophylactic treatment, oligonucleotides areadministered in accordance with the prior invention. Oligonucleotidesmay be formulated in a pharmaceutical composition, which may includecarriers, thickeners, diluents, buffers, preservatives, surface activeagents and the like, in addition to the oligonucleotide. Pharmaceuticalcompositions may also include one or more active ingredients such asantimicrobial agents, anti-inflammatory agents, anesthetics, and thelike in addition to oligonucleotides. Conventional chemotherapeuticagents may also be included.

In one embodiment, the oligonucleotides of the invention are included ina delivery complex. The delivery complex can include the oligonucleotideof the invention and a targeting means. Any suitable targeting means canbe used. For example, the oligonucleotide of the invention can beassociated with (e.g., ionically or covalently bound to, or encapsulatedwithin) a targeting means (e.g., a molecule that results in higheraffinity binding to a target cell, such as a B cell). A variety ofcoupling or cross-linking agents can be used to form the deliverycomplex, such as protein A, carbodiamide, andN-succinimidyl-3-(2-pyridyl-dithio) propionate (SPDP). The complex issufficiently stable in vivo to prevent significant uncoupling prior todelivery to the target cell. In one embodiment, the delivery complex iscleavable such that the oligodeoxynucleotide is released in a functionalform at the target cells.

Dosing is dependent on severity and responsiveness of the condition tobe treated. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual oligonucleotides,and can generally be calculated based on in vitro and in vivo animalstudies. Thus, in the context of this invention, by “therapeuticallyeffective amount” is meant the amount of the compound required to have atherapeutic effect on the treated mammal. This amount, which will beapparent to the skilled artisan, will depend upon the type of mammal,the age and weight of the mammal, the type of disease to be treated,perhaps even the gender of the mammal, and other factors which areroutinely taken into consideration when treating a mammal with adisease. A therapeutic effect is assessed in the mammal by measuring theeffect of the compound on the disease state in the animal. For example,in mammals being treated for cancer, therapeutic effects are assessed bymeasuring the rate of growth or the size of the tumor, or by measuringthe production of compounds such as cytokines, which production is anindication of the progress or regression of the tumor.

Some of the fundamental principles of prodrug approach were discussed inthe previous application.

A “prodrug” is a moiety of the oligonucleotide that can be hydrolyzed toform a purine or pyrimidine antimetabolite. In some embodiments, theantimetabolite is selected from the group consisting of 2′-deoxy,2′,2′-difluorcytidine, 2′-deoxy-3′-thiacytidine,3′-azido-3′-deoxythymidine, 2′,3′-dideoxycytidine,2′,3′-didehydro-3′-deoxythymidine, 2′,3′-dideoxyinosine,5-fluoro-2′-deoxy uridine, 2-fluoro-9-b-D-arabinofuranosyladenine,1-B-D-arabinofuranosylcytosine, 5-azacytidine, 5-aza-2′-deoxycytidine,6-mercaptopurineribo side, 2-chlorodeoxyadeno sine, and pentostatin. Toobtain the antimetabolite, the linkage(s) between the prodrug and othernucleotide(s) of the oligonucleotide is hydrolyzed by natural ornon-naturally occurring enzymes to obtain an antimetabolite nucleoside.In additional embodiments, hydroxyl-protecting groups (e.g.,dimethoxytritryl, monomethoxytrityl, trimethoxytrityl, 9-fluorenylcarbonyl, tetrahydropyranyl, benzoyl, phenoxyacetyl, acetyl, propyl,butyryl, isobutyryl, or other higher homologs) also need to be removedby a deblocking agent in order to obtain the antimetabolite. In someembodiments, amine-protecting groups (e.g., benzoyl, acetyl, propyl,butyryl, isobutryl, phenoxy acetyl, substituted phenoxy acetyl,9-fluorenyl carbonyl, also need to be removed by hydrolysis to obtainthe antimetabolite. Antimetabolites kill both cancer and non-cancerouscells at about the same rate. The prior invention was based on thefinding that oligonucleotides that include at least two CpG moieties andat least one prodrug of an antimetabolite preferentially kill cancerouscells. The oligonucleotide may have one or more ribonucleotides and/orone or more deoxyribonucleotides.

It was noted in the previous application that the oligonucleotides ofthe invention may also have modifications that are not normally found innature or that are found in nature in small quantities. A number ofembodiments were provided for the invention where oligonucleotideshaving at least one nucleotide having a 2′-o-alkyl modification on thesugar of at least one nucleotide. Examples of alkyls include methyl,ethyl, propyl, ethenyl, and higher homologs. Preferably, the homolog hasno more than 6 carbon atoms. Most preferably, the alkyl is methyl. Inanother embodiment, the invention provides an oligonucleotide having atleast one 2′-N-alkyl modification. In yet another embodiment, theinvention provides an oligonucleotide having at least one 2′-halogenmodification.

In one embodiment it was noted that the prodrug can be attached to theoligonucleotide by a 3′-3′ linkage. In another embodiment, the prodrugwas shown to attach to the oligonucleotide by a 5′-5′ linkage. Thesetypes of chemical modifications are known to the skilled person and aredescribed, for example, in M. Koga et al., J. Org. Chem. 56:3757, 1991,EP 0 464 638, and EP 0 593 901, U.S. Pat. No. 5,750,669, each of whichis incorporated herein by reference. The synthesis of an oligonucleotidehaving a 3′-3′ linkage at the 3′ end can be achieved by attaching the 5′end of a nucleoside attached to a solid support; this nucleoside thenwill allow growth of an oligonucleotide from the 3′ end. Similarly, thesynthesis of an oligonucleotide having a nucleotide linked by a 5′-5′linkage at the 5′ end of the oligonucleotide can be achieved by using anucleotide having a support attached at the 3′ end that will allowgrowth of the oligo from the 5′-end.

It was also discussed that the prodrug may also be linked by a 3′-5′linkage or a 5′-3′ linkage, both of which are well known to the skilledartisan. Attachment of a prodrug to the oligo via a linker, will causeliberation of the prodrug at the cancer cell sites by hydrolytic enzymesand will have the effect of the prodrug as well as the CGn*CGoligonucleotide. An example of a point of cleavage between a prodrug andthe oligonucleotide is an ester linkage. The aliphatic esters are chosenfor this purpose, since they are stable, yet can be easily hydrolyzedinside cells by intracellular esterases. Aliphatic phosphate esters,alkyl substituted phosphate esters, and amidates are also part of thisdiscovery, since they are also hydrolyzed by intracellular enzymes.Aliphatic amide linkages are chosen at the other side of linkage. Theamide linkage is generally required in order to attach the prodrug andoligonucleotide. Thus, one end bears a carboxylic or activated ester ofcarboxylic acid, and the other end has a free amino function, to effectthe joining of two moieties. This approach is extensively used inoligonucleotide labeling with various chromophores and ligands (See, P.S. Nelson, M. Kent and Sylvester Muthini, Nucleic Acids Research, Vol.20, No. 23: 6253-6259, 1992; Misiura, K., Durrant, I., Evans, M. R., andGait, M., Nucleic Acids Research, Vol., 18: 4345-4354, 1990; Zendegui,J. G., Vasquez, K. M., Tinsley, J. H., Kessler, D. J. and Hogan, M. E.,Nucleic Acids Research, Vol. 20: 307-314, 1992. Each of these isincorporated herein by reference.).

Exemplary oligonucleotides within the scope of the invention weredepicted in Formulas I-XI, reproduced below. Each of these includes atleast two CpG motifs. These oligonucleotides do not limit the scope ofthe invention. In some embodiments, the prodrug replaces one of thenucleotides of one of the CpG motifs. In other embodiments, theantimetabolite prodrug of Gemcitabine does not replace one of thenucleotides of the CpG motifs.

Each of these formulas shows oligonucleotides that include the prodrugof the antimetabolite 2′-deoxy, 2′,2′-difluorocytidine which has beenchemically modified so that the antimetabolite may offer increasedbiological potency and enhanced structure activity relationship,improved diseased recognition. Oligonucleotides with prodrugs for otherantimetabolites may be used within the scope of the invention may beused in the same way and position as shown for 2′-deoxy,2′,2′-difluorocytidine or in other positions. Intermediates useful inthe synthesis of these oligonucleotides are described below as well.Oligonucleotides of the invention may further include one or moreprotecting groups as defined herein.

For Formulas I through XI below, the subsequent symbols used have thefollowing meaning: C represents cytosine or a modified cytosineincluding: a 5-alkyl cytosine such as 5-methyl cytosine, a 5-alkenylhomolog, a 5-alkynyl homolog, or a 5-halogen analog. The nucleotide withthe C base is the prodrug for 2′-deoxy, 2′,2′-difluorocytidine. Inanother embodiment, the nucleotide with the C base is an analog of2′-deoxy, 2′,2′-difluorocytidine.

The phosphodiester bond is selected from a natural phosphodiester,alkoxy phosphotriester containing a lower alkoxy containing 1 to 6carbon atoms such as OCH₃, OC₂H₅, n-C₃ H₇, iso-C₃H₇, a substituted loweralkoxy, such as OCH₃, OC₂H₅, phosphorothioate, a straight or branchedC-1 to C-6 alkyl, and phosphoramidate. Other internucleotide linkagesmay be used within the context of the prior invention.

In the formula's it was noted that B, B′ and B″ were the same ordifferent natural or modified bases. Natural bases include adenine,cytosine, guanine, thymine, inosine, and uridine. Modified bases include5-methylcytosine, 5-azacytosine, 5-halogen substituted (F, Cl, Br, I)uracil or cytosine, and 5-alkyl substituted uracil or cytosine, such as,C-5 propyne uracil and C-5 propyne cytosine. Purine modifications caninclude 7-deazaadenine, 7-deazaguanine, 7-iodo-7-deaza adenine,7-iodo-7-deazaguanine, 7-propyne-7-deazaadenine,7-propyne-7-deazaguanine. Other bases are well known to the skilledartisan. The repeating portion of the oligonucleotide may have the sameor different bases, and the term B′ should not be construed to implythat the same nucleotide is repeated n number of times, but rather thatthere are n nucleotides each of which has a base that is within thedefinition of B′.

The number n was noted to be between 2 to 50.

S stands for fluorine, chlorine, a sulfur derivative (e.g., S-alkyl), ora nitrogen with alkyl groups (e.g., N—R′, R″, in which R′ and R″ are thesame or different alkyl groups with up to 8 carbon atoms).

In one embodiment (Formula I), the prodrug was shown to attach at the3′-end of an oligonucleotide via a 3′-5′ linkage.

In another embodiment, the invention provided the oligonucleotide shownin Formula II. In this case, the prodrug was shown to be attached at the5′-end of the oligonucleotide via a 5′-5′-Linkage. The oligonucleotidecan be synthesized with a prodrug phosphoramidite, in which thephosphoramidite is at the 5′ of the oligonucleotide.

In another embodiment, the invention provided an oligonucleotide of thefollowing formula:

The prodrug was shown attached at the 5′-end of the oligonucleotide viaa 3′-5′-linkage. In another embodiment, the invention provided anoligonucleotide of the following formula:

In yet another embodiment the invention provided the oligonucleotideshown in Formula V.

In this embodiment, the prodrug was envisaged to be at an internalposition of the oligonucleotide. It was noted that the synthesis of suchan oligonucleotide can be achieved by the use of a phosphoramiditenucleotide of the prodrug using well-known oligonucleotide synthesistechniques.

The invention further provided an oligonucleotide having the followingformula (VI) :

In this embodiment, the prodrug was shown to be attached to the 3′-endof an oligonucleotide via a lipophilic ester group at the prodrug's5′-position. “X, Y=” means that X and Y are independently chosen fromthe three groups shown. The coupling could be done with an amino linkeroligonucleotide. The amino linker oligonucleotides having various spacerlengths are well known in the oligonucleotide chemistry. (See, P. S.Nelson, M. Kent, S. Muthini, Nucleic Acids Research, Vol. 20, No. 23:6253-6259, 1992; F. Berg, D. Praseuth, A. Zerial, N. Thoung, U.Asseline, T. Le Doan, C. Helene, Nucleic Acids Research, 18: 2901-2908,1990, which are hereby incorporated by reference.)

Suitable ester groups include carboxylic ester, methylene and phosphateester groups. The assembly of oligonucleotides having a lipophilic esteris well known to the skilled artisan. For example, if the prodrug is2′-deoxy, 2′,2′-difluorocytidine, the oligonucleotide can be synthesizedusing the 2,2′-difluorocytidineactive ester, an example of which isshown in formula XVIII. The oligonucleotide in this case will have a3′-amino linker, which is well known in the art. The finaloligonucleotide as depicted in Formula VII will be formed from thereaction of 3′-amino linker oligonucleotide and the active ester offormula (XVII).

In another embodiment, the invention provides an oligonucleotide of thefollowing formula:

In this oligonucleotide, the prodrug was shown to be attached via alinker, at its 3′-position to the 5′-end of an oligonucleotide. Thesynthesis of oligonucleotides having a lipophilic group can beaccomplished by procedures cited in the literature, e.g., (Nikolai N.Polushin and Jack Cohen, Nucl. Acids Res., 22: 5492-5496, 1995). Onesuch procedure will require the prodrug attached to solid support at the5′-end, which will subsequently be treated with an amino linker at the3′-end. The 3′-amino linker prodrug can then be coupled with anoligonucleotide having a carboxylic linker at its 5′-end. The generalsynthesis of oligonucleotide having a 5′-aliphatic carboxylic group canbe derived from commercially available products, such asDMT-Thymidine-succinyl hexamide amidite, which is available fromChemGenes Corporation, Wilmington, Mass., catalog item number CLP-2244.

In yet another embodiment, the invention provided an oligonucleotide ofthe following formula (VIII):

In this embodiment, the prodrug was shown to be attached at the 3′ endof RNA or a 2′-modified RNA oligonucleotide, via a 3′-3′ linkage. The Xstands for H, methyl, ethyl, a higher C3-C6 alkyl homolog, a C2-C6alkenyl, a C2-C6 straight or branched alkynyl, an amino C1-C6 alkyl, anamino C2-C6 alkenyl, cyclopropyl, an allyl, a C1-C6 alkynylalkoxy, or anaminoalkoxy.

In yet another embodiment, the invention provided oligonucleotides ofthe following formula (IX):

Where X was defined as used for Formula VII. Such oligonucleotides havea prodrug attached via a 5′-5′linkage at the 5′ end of RNA or a2′-modified RNA. The attachment of a prod rug via its 5′-end can beachieved using a prodrug phosphoramidite, for example, the prodrug thatis depicted in formula XX. The X is used as defined in reference toFormula VIII.

In yet another embodiment the invention provided an oligonucleotide ofthe following formula (X):

In this embodiment, the prodrug is attached at its 3′ end to the 3′-endof RNA or a 2′-modified RNA oligonucleotide. The attachment of theprodrug at its 3′-end can be achieved using the prodrug bound to a solidsupport at its 5′-end, as depicted in formula XV for the prodrug of2′-deoxy,2′,2′-difluorocytidine.

In yet another embodiment, the invention provides an oligonucleotide ofthe following formula:

Here, the prodrug is attached at its 3′ end to the 5′-end of RNA or a2′-modified RNA.

Oligonucleotides can be synthesized de novo using any of a number ofprocedures well known in the art. For example, the 0-cyanoethylphosphoramidite method (S. L. Beaucage and M. H. Caruthers, Tet. Let.22:1859, 1981; U.S. Pat. Nos. 4,415,732 and 4,458,066, (Caruthers), andU.S. Pat. No. Re 34,069, (Koster)) the nucleoside H-phosphonate method(Garegg et al., Tet. Let. 27: 4051-4054, 1986; Froehler et al., Nucl.Acid. Res 14: 5399-5407, 1986; Garegg eg al., Tet. Let. 27: 4055-4058,1986; Gaffney et al., Tet. Let. 29: 2619-2622, 1988) can be used tosynthesize oligonucleotides of the invention. Each of the abovereferences is hereby incorporated by reference. These chemistries can beperformed by a variety of automated oligonucleotide synthesizersavailable in the market. The oligonucleotides used in accordance withthis invention may be conveniently and routinely made through thewell-known technique of solid phase synthesis. Equipment for suchsynthesis is sold by several vendors including Applied Biosystems(Foster City, Calif.). It is also well known to use similar techniquesto prepare other oligonucleotides such as phosphorothioates or alkylatedderivatives. It is also well known to use similar techniques andcommercially available modified phosphoramidites and solid supports,such as polystyrene, various silica gels in beads or powder forms, andcontrolled-pore glass (CPG) products to synthesize naturally-occurringand modified oligonucleotides. An example of use of a solid support tosynthesize oligonucleotides is provided in U.S. Pat. No. 6,646,118 whichis incorporated herein by reference.

SUMMARY OF THE INVENTION

The present application pertains to chemical compounds that aremodifications of Gemcitabine which provide improved biochemical andbiological effectiveness of the cancer fighting property of Gemcitabine.

Similar to the prior invention in one embodiment, the oligonucleotide ofthe invention has at least one nucleotide having a ribose sugar moiety.In another embodiment, the oligonucleotide of the invention has at leastone nucleotide having a 2′-deoxyribose sugar moiety. In yet anotherembodiment, the oligonucleotide has at least one 2′-o-alkyl nucleotide,2′-n-alkyl nucleotide, or 2′-O-halogen nucleotide, wherein the alkyl hasbetween about 1 and about 6 carbon atoms. Nucleotides of theoligonucleotides are connected by covalent internucleoside linkages.Examples of covalent internucleoside linkages include phosphodiesterlinkages, C1-C6 alkoxy phosphotriester linkages, phosphorothioatelinkages and phosphoramidate linkages. In some embodiments, the prodrugis attached to at least one of the multiple nucleotides by a linker ofthe following formula: 2

Linker;

The invention further provides a pharmaceutical composition thatincludes a therapeutically effective amount of any of theoligonucleotides disclosed herein. In some embodiments, thepharmaceutically acceptable carrier is lipofectin.

The invention further provides a compound having purity in excess of 98%by HPLC, and having the following formula;

Wherein R is selected from the group consisting of H, a C1-C6 alkyl, ahalogen, a C2-C6 alkenyl, and a C2-C6 alkynyl;

x is an amine-protecting group that is stable in oligonucleotidesynthesis conditions; and

y, and z are each selected from H, a hydroxyl-protecting group that isstable in oligonucleotide synthesis conditions and a group that can beattached to a solid support. In some embodiments, the compound groupthat is attachable to a solid support has the formula C(O)-M-C(O)—NH,where M is selected from the group consisting of succinyl, oxalyl, andhydroquinolynyl, and wherein the Spacer is a C1-C20 alkyl,ethyloxyglycol, or a combination of alkyl and ethyleneglycoxy, and theSpacer is attached to the solid support.

Inter alia, the present application also discloses a compoundrepresented by the formula

Wherein R is selected from the group consisting of H, a C1-C6 alkyl, ahalogen, a C2-C6 alkenyl, and a C2-C6 alkynyl;

x is an amine-protecting group that is stable in oligonucleotidesynthesis conditions;

z is a hydroxyl-protecting group that is stable in oligonucleotidesynthesis conditions; and, in some embodiments, the compound group thatis attachable to solid support has the formula C(O)-M-C(O)—NH, where Mis selected from the group consisting of succinyl, oxalyl, andhydroquinolynyl, and wherein the Spacer is a C1-C20 alkyl,ethyloxyglycol, or a combination of alkyl and ethyleneglycoxy, and theSpacer is attached to the solid support.

Other modifications of the compounds and the requirements of the puritylevels of the disclosed compounds are discussed further in the detaileddescription below.

DETAILED DESCRIPTION OF THE INVENTION

Similar to the invention in the parent application the present inventionrelates to CpG oligonucleotides having at least one modifiedGemcitabine, a nucleoside antimetabolite which can be classified as a“prodrug”. Various modifications were envisaged and disclosed for theGemcitabine nucleoside in order to modulate biochemical and biologicalproperties and to develop ideal therapeutic candidates. The presentinvention addresses the chemical modifications which are consideredgermane to this approach. The detailed descriptions of representativestructures are illustrated diagrammatically in the Background section inFormulas I-XI.

Various embodiments are briefly summarized in the following paragraphs.

In one embodiment, the oligonucleotide of the invention has at least onenucleotide having a 2′-deoxyribose sugar moiety. In yet anotherembodiment, the oligonucleotide has at least one 2′-o-alkyl nucleotide,2′-n-alkyl nucleotide, or 2′-halogen nucleotide, wherein the alkyl hasbetween 1 and 6 carbon atoms. Nucleosides of the oligonucleotides areconnected by covalent internucleotide linkages. Examples of covalentinternucleotide linkages include phosphodiester linkages, C1-C6 alkoxyphosphotriester linkages, phosphorothioate linkages and phosphoramidatelinkages. In some embodiments, the prodrug is attached to at least oneof the multiple nucleotides by a linker described by the following classof formulas.

Description of Linkers:

A linker may have a formula illustrated below in formula (XII);

Aliphatic amide linkages are chosen at the other side of linkage of themodified nucleoside antimetabolite, while one end is attached tooligonucleotide chain. The amide linkage is generally required in orderto attach the prodrug and oligonucleotide. Thus, one end bears acarboxylic or activated ester of carboxylic acid, and the other end hasa free amino function, to effect the joining of two moieties.

The coupling could be done with an amino linker oligonucleotide. Theamino linker oligonucleotides having various spacer lengths are wellknown in the oligonucleotide chemistry.

Suitable ester groups include carboxylic ester and phosphate estergroups.

For instance, the modified antimetabolite prodrug is attached to the3′-end of an oligonucleotide via a lipophilic ester group at theprodrug's 5′-position. “X, Y═” in Formula XII means that X and Y areindependently chosen from the three groups shown.

The modified antimetabolite prodrug may also be linked by a 3′-5′linkage or a 5′-3′ linkage.

The general synthesis of oligonucleotide having a 5′-aliphaticcarboxylic group can be derived from commercially available products,such as DMT-thymidine-succinyl hexamide amidite, which is available fromChemGenes Corporation, Wilmington, Mass., catalog item number CLP-2244.

The invention also provides a pharmaceutical composition that includes atherapeutically effective amount of any of the oligonucleotidesdisclosed. In some embodiments, the pharmaceutically acceptable carrieris lipofectin.

The invention provides generic compounds having purity in excess of 97%by HPLC, and having the following formulas XIV and XV; and furtherprovides the compounds having purity in excess of 98% by HPLC.

Further embodiments relate to 5′-OH protected derivatives of 2′-deoxy,2′,2′-difluorocytidine or of the 4-amine protected 2′-deoxy,2′,2′-difluorocytidine. These are useful in intermediates for thepreparation of 2′-deoxy, 2′,2′-difluorocytidine attachedoligonucleotides.

Wherein, R is selected from the group consisting of H, a C1-C6 alkyl, ahalogen, a C2-C6 alkenyl, and a C2-C6 alkynyl;

x is an amine-protecting group that is stable in oligonucleotidesynthesis conditions; and y, and z are each selected from the groupconsisting of (H, a hydroxyl-protecting group that is stable inoligonucleotide synthesis conditions, phosphoramidites, and a group thatcan be attached to a solid support).

In some cases y is hydroxyl-protecting group such as DMT(dimethoxytritryl), MMT (monomethoxytrityl), TMT (trimethoxytrityl),FMOC (9-fluorenyl carbonyl chloride), tetrahydropyranyl, benzoyl,phenoxyacetyl, acetyl, propyryl, butyryl, isobutyryl, or other higherhomologs.

The amine-protecting group x for the exocyclic amine as an amide bondcould further comprise one or more of the following: a lower (i.e.,C1-C6) alkanoyl group containing a straight or a branched chain alkylgroup as defined above, an aryl or substituted aryl having a C1-C6 alkylor halogen as a substituent on the aryl ring, a phenoxy acetyl orappropriately protected phenoxy acetyl for fast deprotection, atrifluroacetyl or FMOC group, an imine derivative such as formamidine ordimethylformamidine. Such protecting groups are required to offer mildand convenient deprotection conditions after the synthesis of theoligonucleotides derived from the compounds of the present invention,and can be cleaved with a suitable reagent to generate free NH2 groupsat the end of the oligonucleotide synthesis.

In some embodiments, the group of spacer compounds used for attachmentto a solid support has the general formula O—C(═O)-M-C(═O)—NH, where Mcan be selected from the group consisting of succinyl, oxalyl, andhydroquinolynyl, a C1-C6 alkyl, ethyloxyglycol, or a combination ofalkyl and ethyleneglycoxy.

The invention further discloses an active ester having the formula:

wherein R, M, x, y and z are as defined above for the active esters asshown by the formula XVII, having a purity level approximately 95% andabove by HPLC.

The invention additionally provides a phosphoramidite having purity inexcess of 97% and 98% by HPLC, as shown by the formula XVIII and XIX.

Wherein, each of y and z is a hydroxyl-protecting group that is stablein oligonucleotide synthesis conditions; x and R are as defined above,and R′ and R″ are independently selected and are either a C1-C6 alkyl ora C2-C6 cycloalkyl.

The sugar and bases of one or more nucleotides that make up theoligonucleotide may be a chimera consisting of ribonucleosides, orribonucleosides in one or more positions. In the case of ribo basesnucleosides may have a 2′-o-alkyl, 2′-n-alkyl, or 2′-halogenmodifications on the sugar. Examples of alkyls include methyl, ethyl,propyl, ethenyl, and higher homologs. Preferably, the homolog has nomore than 6 carbon atoms. Most preferably, the alkyl is methyl. In suchmodification the oligonucleotide may have at least one nucleotide havingat least one 2′-n-alkyl modification. Similarly, such Oligonucleotidemay have at least one nucleoside having at least one 2′-halogenmodification.

Oligonucleotides based on the present invention may include one or moreprotecting groups for stability during oligonucleotide delivery or invivo conditions.

Further, the oligonucleotides can be obtained from existing nucleic acidsources (e.g. genomic or cDNA), but are preferably synthetic, and have adefined sequence (e.g. produced by oligonucleotide synthesis).

The sugar hydroxyl-protecting groups can be selected from the group(dimethoxytritryl, monomethoxytrityl, trimethoxytrityl, 9-fluorenylcarbonyl, tetrahydropyranyl, benzoyl, phenoxyacetyl, acetyl, propyl,butyryl, isobutyryl, or other higher homologs); these groups also needto be removed by a deblocking agent in order to obtain theoligonucleotides incorporating modified antimetabolites.

This invention is based on the finding that oligonucleotides thatinclude at least two CpG moieties and at least one prodrug of anantimetabolite or modified antimetabolite preferentially kill cancerouscells. The oligonucleotide may have one or more ribonucleotides and/orone or more deoxyribonucleotides.

The modified antimetabolite prodrug can be attached to theoligonucleotide by a 3′-3′ linkage, or by a 5′-5′ linkage, or by a 3′-5′or a 5′-3′ linkage. These chemical modifications are well known in theart. See M. Koga et al., J. Org. Chem. 56:3757, 1991, EP 0 464 638, andEP 0 593 901, U.S. Pat. No. 5,750,669.

In another embodiment the oligonucleotide of this invention, the prodrugis attached via a linker, at its 3′-position to the 5′-end of anoligonucleotide. The synthesis of oligonucleotides having a lipophilicgroup can be accomplished by procedures cited in the literature, e.g.,(Nikolai N. Polushin and Jack Cohen, Nucl. Acids Res., 22: 5492-5496,1995). One such procedure will require the prodrug attached to solidsupport at the 5′-end, which will subsequently be treated with an aminolinker at the 3′-end. The 3′-amino linker prodrug can then be coupledwith an oligonucleotide having a carboxylic linker at its 5′-end.

In one embodiment the invention relates to exocyclic amine protectedderivatives of 2′-deoxy, 2′,2′-difluorocytidine which are shown inFormula XX, which is an intermediate for the preparation of various2′-deoxy, 2′,2′-difluorocytidine attached oligonucleotides.

Yet another embodiment relates to 3′- or 5′-phosphoramidite derivativesof 2′-deoxy-2′,2′-difluorocytidine (formulas XIV and XV), preferablysynthesized with the phosphorylating reagent2-cyanoethyl-n,n-diisopropyl-amino phosphoramidite. Alternately, thephosphorylating reagent methoxy-n,n-diisopropylaminophosphinylphosphoramidites may be used in the place of the2-cyanoethyl-n,n-diisopropylamino phosphoramidite. These compounds havepurity exceeding 97% and have coupling efficiency of greater than 98% inless than 100 seconds under standard DNA/RNA synthesis couplingconditions. The standard coupling conditions are outlined in the DNA/RNAsynthesizer manual of MerMade Instrument. The coupling efficiency wasmonitored by carrying out oligo synthesis in the instrument model,Expedite 8909, and using the built in per step coupling monitor.

1. A compound having purity in excess of 97% by HPLC, having theformula:

wherein R is selected from the group consisting of H, a C1-C6 alkyl, ahalogen, a C2-C6 alkenyl, and a C2-C6 alkynyl; x is an exocyclicamine-protecting group that is a member of the group ((C1-C6) alkanoylgroup containing a straight or a branched chain alkyl group, an aryl orsubstituted aryl having a C1-C6 alkyl or halogen as a substituent on thearyl ring, phenoxy acetyl or appropriately protected phenoxy acetyl,trifluroacetyl, FMOC group, imine derivative, formamidine,dimethylformamidine, and dialkylformamidine); and y, and z are eachselected from the group (H, a hydroxyl-protecting group that is stableduring oligonucleotide synthesis, phosphoramidite, and a group that canbe attached to a solid support).
 2. A compound having purity in excessof 97% by HPLC, having the formula:

wherein R is selected from the group consisting of (H, C1-C6 alkyl,halogen, C2-C6 alkenyl, and C2-C6 alkynyl); x is an exocyclicamine-protecting group that is a member of the group ((C1-C6) alkanoylgroup containing a straight or a branched chain alkyl group, an aryl orsubstituted aryl having a C1-C6 alkyl or halogen as a substituent on thearyl ring, phenoxy acetyl or appropriately protected phenoxy acetyl,trifluroacetyl, FMOC group, imine derivative, formamidine,dimethylformamidine, and dialkylformamidine); and y, and z are eachselected from the group (H, a hydroxyl-protecting group that is stableduring oligonucleotide synthesis, phosphoramidite, an active ester, anda group that can be attached to a solid support).
 3. The compound ofclaim 1 or 2 having purity in excess of 98% by HPLC.
 4. The compound ofclaim 1, wherein z is C(O)-M-C(O)—NH, where NH is attached to a solidsupport, where M is selected from the group consisting of (succinyl,oxalyl, hydroquinolynyl, C1-C20 alkyl, ethyloxyglycol, and a combinationof alkyl and ethyleneglycoxy).
 5. The compound of claim 2, wherein z isC(O)-M-C(O)—NH, where NH is attached to a solid support, M is selectedfrom the group consisting of (succinyl, oxalyl, hydroquinolynyl, C1-C20alkyl, ethyloxyglycol, and a combination of alkyl and ethyleneglycoxy).6. The compound of claim 1, where said active ester z has the structure:

wherein M is selected from the group consisting of (succinyl, oxalyl,hydroquinolynyl, C1-C20 alkyl, ethyloxyglycol, and a combination ofalkyl and ethyleneglycoxy).
 7. A compound of claim 2, where said activeester z has the formula:

wherein M is selected from the group consisting of (succinyl, oxalyl,hydroquinolynyl, C1-C20 alkyl, ethyloxyglycol, and a combination ofalkyl and ethyleneglycoxy).
 8. The compound of claim 2 where saidphosphoramidite z has the formula:

and R′ and R″ are independently selected from the group consisting of aC1-C6 alkyl and a C2-C6 cycloalkyl.
 9. A compound of claim 1, where saidphosphoramidite z has the formula:

and R′ and R″ are independently selected from the group consisting of aC1-C6 alkyl and a C2-C6 cycloalkyl.
 10. The compound of claim 8 or 9having purity in excess of 98% by HPLC.